Many biologically active proteins or polypeptides are produced in eukaryotic cells but are found in only minute quantities in their native cells or tissues. These proteins can be extremely difficult or expensive to purify in quantity, often due to the scarcity of the natural supply. Recombinant DNA methods have allowed the generation of complementary DNA (cDNA) sequences which code for the proteins of interest. These cDNAs can be inserted into cloning vectors (along with the appropriate regulatory regions necessary for expression) and introduced into suitable host cells. The introduction of such vectors into the appropriate cells allows the cells to produce the polypeptide encoded by the cDNA. Similar vectors can be made using the genomic (ie with introns and exons) sequences which make up the gene of interest. Eukaryotic cells (and in particular mammalian cells) are able to perform a number of post-translational modifications (such as amidation, glycosylation etc) which are not observed with expression in prokaryotic cells. Consequently, mammalian cells often produce proteins which resemble more closely the natural, biologically active protein molecules. The large scale culture of mammalian cells, however, is expensive and slow compared to the culture of prokaryotic cells, and methods are continually sought to increase the productivity per animal cell and to decrease the time to generate useful quantities of the protein product by the animal cell.
In general, the introduction of foreign DNA (such as the vectors described above) into animal cells leads to the random integration of one or more copies of the DNA into the genome of the cell. The level of gene expression from the foreign gene is highly dependent on the position of integration of the foreign DNA into the host genome (`position effects`). Integration into so-called `active regions` usually produces somewhat higher levels of expression but these are often still low when compared to the levels seen with a native chromosomal (non-transfected) gene. The levels of the transfected gene do not generally correlate with the copy number in any one cell; another consequence of position effects.
A common technique used to increase the productivity of transfected host cells is the in vivo amplification of the integrated foreign DNA to produce high copy-number integrants. In these procedures, the gene of interest (along with appropriate control sequences) is co-transfected with a gene which can have a protective effect against a toxic substance. A commonly used protective gene is the dihydrofolate reductase (DHFR) gene. When increasing concentrations of methotrexate (MTX), a competitive inhibitor of the essential enzyme DHFR, is applied to the transfected cells, only cells with higher expression levels of DHFR will survive. As MTX levels are increased further, only cells which amplify the copy number of the DHFR gene (and consequently the co-transfected recombinant expression construct) will survive. In this way, the copy number and hence expression levels (ie productivity per cell) of the cDNA can be increased.
Recently, a novel enhancer-like element from the human globin locus has been described (Grosveld et al., 1987; WO 89/01517) which directs high level, position independent, copy-number dependent expression of heterologous genes in erythroid cells. This element is extremely cell-type specific and only functions in erythroid cells. This dominant control region (DCR) element has been used to overcome position effects in the expression of human .beta.-globin in erythroid cells. This reference, WO 89/01517, describes the use of this enhancer element in the expression of .beta.-globin from the globin promoter.
However, the use of the DCRs for expression of heterologous proteins has not become widespread because of the nature of erythroid cells and the perception that the cells are incapable of significant levels of secretion.
There is a need for a mammalian expression system which is capable of expressing heterologous polypeptides.
There is also a need for improved mammalian expression systems which are capable of expressing polypeptides at high levels.
There is also a need for a mammalian expression system which is capable of expressing a polypeptide at high levels and secreting the polypeptide expressed.